Anti gadph antibody

The treated cells were mechanically homogenized and centrifuged at 3000 g for 30 min at 4°C to collect supernatant. The protein extracts were denatured and were heated for 10 min at 98°C. The samples (40 μg/sample) were then loaded on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) for electrophoresis and transferred to a PVDF membrane. The membrane was incubated with blocking solution for 1 hr and probed with the following primary antibodies: anti-α₂-AR (Antibodies.com), anti-iNOS, anti-Arg1, anti-Ym1 (Abcam), anti-PPAR (this antibody and below were all from Cell Signaling Technology), anti-Akt, anti-p-Akt (Ser473), anti-p-PTEN (Ser380), anti-PTEN, anti-p-GSK-3β (Ser9), anti-p-PDK (Ser241), anti-STAT6, and anti-IRF-4 in 1:1000 dilution overnight at 4°C, followed by HRP-conjugated secondary antibody for 1 hr. The loading control was probed with anti-GADPH antibody (1:10,000, Sigma-Aldrich). The membrane was washed with TBST and visualized with an enhanced chemiluminescence (ECL) system (Santa Cruz, UK). The protein bands were captured with an image processor (GeneSnap; SYNGENE^®, Cambridge, UK).